The goal of these studies is to investigate the role of phase separations and resultant defect sites in membranes as a potential mechanism through which ethanol exerts its effects on biomembranes. Studies will be carried out in model phospholipid membranes with varying degrees of phase separation obtained by varying the phospholipid composition. Mixtures of dipalmitoylphosphatidylcholine and dilauroylphosphatidylcholine will be used. A series of 1-alkanols from ethanol to octanol will be examined to define better the physical forces driving ethanol-membrane interactions. Macroscopic partition coefficients will be measured to determine total membrane concentration of alcohol as a function of phase separation. The effects of alcohols on lateral and vertical organization of the phospholipid bilayer will be investigated as a function of phase separation utilizing fluorescence probes. Phospholipase A2 activity will be employed as a probe of the defect regions in membranes between phase separated domains. The effects of alcohols on phospholipasee A2 activity will be investigated in the model membranes as a function of the degree of phase separation. The proposed research will provide a thorough examination of the role of phase separation and defect sites in the ability of ethanol to enter and perturb model phospholipid membranes and will provide a basis for extension of similar studies to biological membranes.